Prebiotics are dietary substances that stimulate growth of selected groups of microorganisms in the colon and in addition may have other health benefits. Galactooligosaccharides (GOS), fructooligosaccharides (FOS), lactulose, and isomaltooligosaccharides (IMO) are among the few well-established prebiotics. In human milk, oligosaccharides constitute the third largest component, present in amounts as much as 20-25 g/l around parturition, later declining to 5-15 g/L. With few exceptions, all known human milk oligosaccharides (HMOs) have a lactose core and are elongated via linkage to one or more units of galactose and N-acetylglucosamine, and can be decorated with several sialic acid and fucose residues. More than 100 different such glycan structures have been identified and approximately 10-20% of these are sialylated (Bode, 2012, Glycobiology 22(9): 1147-1162). Sialylation and/or fucosylation of many of these HMOs appear to convey important functional properties. For example, HMOs can bind human pathogens, such as, Escherichia coli K1, Haemophilus influenzae, Pasteurella multocida, Neisseria meningitidis, Campylobacter jejuni, Vibrio cholerae, Helicobacter pylori and Streptococcus agalactiae and thereby reduce the incidence of diarrhoea and other diseases in infants. This ability of HMOs to function as soluble decoy receptors for human pathogens is most likely enhanced by their diversity, since mannose-containing glycoproteins, sialylated and fucosylated glycans each target different subsets of pathogens (Kunz et al., 2000, Ann. Rev. Nutrition 20:699-722). In addition, sialylated HMOs may modulate the immune system; for example T cell cytokine production is stimulated by sialylated HMOs in vitro (Eiwegger et al., 2004, Pediatric Rev. 56:536-540). In most cases, the active HMO molecules have not been identified, but in the case of necrotising enterocolitis, a frequent and often fatal disease in infants, the protective effect was recently shown to be due to a single molecule, disialyllacto-N-tetraose, using a rat model (Jantscher-Krenn et al., 2012, Gut 61:1417-1425).
Bovine milk, which forms the basis for most infant formula, has a very low oligosaccharide content when compared with human milk, with a different sialylation and fucosylation profile. In an attempt to mimic the composition of human milk, milk formula is currently supplemented with (non-HMO) GOS and FOS. However, due to their lack of sialic acid residues, the added GOS and FOS are unlikely to provide the therapeutic benefits of HMOs, described above (Bode, 2012, supra).
Efforts to sialylate GOS and FOS rely on glycan sialylation, which can be achieved chemically as well as enzymatically using different types of enzymes [1]. For example, a trans-sialidase enzyme (TcTS) from Trypansoma cruzi, the causative agent of Chagas disease, has been used to transfer sialic acid from a donor to an acceptor glycan [2]. However, in the context of industrial production of food-grade HMOs, the T. cruzi trans-sialidase has a major drawback, namely that it constitutes an important virulence factor within T. cruzi [3].
A native sialidase (TrSA) found in the non-pathogenic Trypansoma rangeli, has been used as a starting point for generating mutant enzymes that possess trans-sialidase activity [4]. Although this sialidase shares 70% sequence identity with that of TcTS, and has the same overall tertiary structure, it is a strict hydrolase having no detectable trans-sialidase activity [4]. The sialidase, TrSA, and the trans-sialidase, TcTS, share a common double displacement mechanism with a tyrosine as catalytic nucleophile [5] [6]. In TcTS, the acceptor binding site consists of Tyr119 and Trp312 forming stacking interactions with the acceptor sugar [7]. In TrSA, Trp313 (corresponding to Trp312 in TcTS) is found in a different conformation due to a Gln at position 284, while it has a Ser residue at position 120 corresponding to Tyr119 in TcTS [8]. In addition to these differences in the acceptor binding site, a conserved Asp96 hydrogen bonds differently to sialic acid in the two enzymes, possibly due to two residue differences, Val96Met and Pro98Ala. Initial attempts based on TrSA single point mutants, failed to generate an enzyme with any trans-sialidase activity. Subsequent studies revealed the need for a combination of 5 point mutations TrSA, comprising Ser120Tyr, Gly249Tyr, and Gln284Pro at the acceptor-binding site as well as Met96Val, and Ala98Pro at the sialic acid binding pocket to confer trans-sialidase activity (1% of TcTS) to TrSA. An additional single mutation Ile37Leu increased the levels of trans-sialidase activity to 10% of a T. cruzi trans-sialidase [4]. Furthermore, kinetic data indicate that these TrSA mutants display a >25-fold lower affinity for lactose and >100-fold higher turnover (kcat) for the undesired, competing hydrolysis compared to TcTS [4] indicating a considerable need for improvement before such an enzyme would have any practical value for. trans-sialylation.
Despite the relatively close sequence homology between TrSA and TcTS, there is no evidence that the native sialidase expressed by Trypansoma rangeli has any trans-sialidase activity. Isolation and expression of a TrSA gene from Trypansoma rangeli is reported by Smith et al [31]. The isolated TrSA gene encodes an inactive protein, likely due to the substitution of a strictly conserved arginine, that functions by coordinating the carboxyl of sialic acid, by a cysteine residue [31]. Smith et al., also submitted a TrSA gene encoding sialidase (Q08672) to GenBank, which is predicted to be an anhydrosialidase [32]. In addition to lacking the Arg residue required for coordinating the carboxyl of sialic acid, this sialidase (Q08672) lacks the mutations S119Y and Q284P that are required to establish the acceptor binding site, and for this reason cannot function as a trans-sialidase.
Buschiazzo et al., [33] report the isolation of a Trypansoma rangeli gene that is predicted to encode a TrSA, UNIPROT: Q08672 having 70% sequence identity to TcTS, which is a common feature of other TrSAs having only hydrolytic activity. One amino acid substitution in the primary sequence of a TrSA, found essential for obtaining a mutant TrSA having measurable trans-sialiase activity is Gly249-Tyr, which decreases hydrolytic activity [4]. A second mutation, Ile-37Leu, which in combination with Tyr120, significantly enhances trans-sialidase activity in this mutant [4]. Neither of these mutations is found in TrSA, UNIPROT: Q08672.
In human milk, lactose or HMOs of various lengths can be sialylated in α2-3 or α2-6 linkage which can be added to a terminal galactose or a subterminal N-acetyl-glucosamine, thereby contributing to the diversity of HMOs present. Efforts to mimic such complex oligosaccharide compositions require a trans-sialidase that can transfer sialic acid to a variety of different acceptor groups. Although it is well established that TcTS can sialylate the terminal galactose of a glycan, there is no documented evidence of a trans-sialidase that can use other acceptor groups, which is essential if the diversity of HMOs is to be obtained synthetically.
Accordingly, there remains a need for an enzyme having trans-sialidase activity, that is neither a virulence factor nor derived from a pathogenic organism; and further has no significant sialidase hydrolytic activity, and that can transfer a sialic acid moiety to a range of different acceptor groups present in a glycan molecule.